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1.
J Proteome Res ; 16(2): 528-537, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-27977922

RESUMO

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of B. pertussis. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in B. pertussis, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of B. pertussis. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines.


Assuntos
Antígenos de Bactérias/biossíntese , Bordetella pertussis/genética , Toxina Pertussis/biossíntese , Vacina contra Coqueluche/biossíntese , Proteoma/análise , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/crescimento & desenvolvimento , Bordetella pertussis/patogenicidade , Cromatografia Líquida , Fermentação , Expressão Gênica , Humanos , Sulfato de Magnésio/farmacologia , Espectrometria de Massas , Toxina Pertussis/antagonistas & inibidores , Toxina Pertussis/genética , Vacina contra Coqueluche/genética , Vacina contra Coqueluche/imunologia , Proteoma/biossíntese , Proteoma/genética , Proteoma/imunologia , Coqueluche/imunologia , Coqueluche/microbiologia , Coqueluche/prevenção & controle
2.
J Proteome Res ; 12(4): 1898-908, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23410224

RESUMO

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.


Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Vacinas Meningocócicas/análise , Vacinas Meningocócicas/química , Proteômica/métodos , Animais , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Citoplasma/química , Detergentes/química , Feminino , Lipoproteínas/análise , Lipoproteínas/química , Vacinas Meningocócicas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis Sorogrupo B/patogenicidade
3.
Anal Chem ; 85(12): 6089-94, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23672432

RESUMO

Using a capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) method, the isoelectric points (pI) of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of inactivated polio vaccines (IPV)-Mahoney (type 1), MEF (type 2), and Saukett (type 3)-as well as for attenuated oral polio vaccines (OPV) and Sabin types 1, 2, and 3. A method for analyzing biological hazardous components (biological safety level 2) was set up for the CIEF-WCID analyzer used. This method is based on closed circuits. The determined pI's were 6.2 for Mahoney, 6.7 for MEF-1, and 5.8 for Saukett. The pI's of Sabin types 1, 2, and 3 viruses were 7.4, 7.2, and 6.3, respectively. Resolution of the virus peaks was shown to be reproducible. Using this adjusted CIEF-WCID technique, the pI of biologically hazardous components like toxins or viruses can be determined, which is beneficial for the development of vaccine production methods among others.


Assuntos
Técnicas Biossensoriais/métodos , Poliovirus/isolamento & purificação , Animais , Chlorocebus aethiops , Eletroforese Capilar/métodos , Células HeLa , Humanos , Focalização Isoelétrica/métodos , Ponto Isoelétrico , Poliovirus/química , Células Vero
4.
Biotechnol Bioeng ; 110(5): 1354-65, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23192424

RESUMO

The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production processes is necessary as the initial process development has been done decades ago. An up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up. This lab-scale version should be representative of the large scale, meaning a scale-down model, to allow experiments for process optimization that can be readily applied. Initially the separate unit operations were scaled-down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large-scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small-scale. Challenges in translating large-scale operations to lab-scale are discussed, and the concessions that needed to be made are described. The current scale-down model for cell and virus culture (2.3-L) presents a feasible model with its production scale counterpart (750-L) when operated at setpoint. Also, the current scale-down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small-scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it is shown that the scale-down unit operations can be used consecutively to prepare trivalent vaccine at lab-scale with comparable characteristics to the product produced at manufacturing scale.


Assuntos
Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Vacina Antipólio de Vírus Inativado/biossíntese , Vacina Antipólio de Vírus Inativado/isolamento & purificação , Animais , Biotecnologia/instrumentação , Técnicas de Cultura de Células/instrumentação , Chlorocebus aethiops , Cromatografia Líquida , Formaldeído/farmacologia , Poliovirus/efeitos dos fármacos , Poliovirus/imunologia , Poliovirus/isolamento & purificação , Poliovirus/fisiologia , Vacina Antipólio de Vírus Inativado/metabolismo , Células Vero , Inativação de Vírus/efeitos dos fármacos
5.
PLoS One ; 18(11): e0291625, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38015925

RESUMO

Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. Additionally, it is critical for establishing the time of infection in bioreactors and monitoring cell culture response to targeted infection over time. However, when cell counting is performed manually, the time involved can become substantial, particularly when multiple cultures need to be handled in parallel. Automated cell counters, which enable significant time reduction, are commercially available but remain relatively expensive. Here, we present a machine learning (ML) model based on YOLOv4 that is able to perform cell counts with a high accuracy (>95%) for Trypan blue-stained insect cells. Images of two distinctly different cell lines, Trichoplusia ni (High FiveTM; Hi5 cells) and Spodoptera frugiperda (Sf9), were used for training, validation, and testing of the model. The ML model yielded F1 scores of 0.97 and 0.96 for alive and dead cells, respectively, which represents a substantially improved performance over that of other cell counters. Furthermore, the ML model is versatile, as an F1 score of 0.96 was also obtained on images of Trypan blue-stained human embryonic kidney (HEK) cells that the model had not been trained on. Our implementation of the ML model comes with a straightforward user interface and can image in batches, which makes it highly suitable for the evaluation of multiple parallel cultures (e.g. in Design of Experiments). Overall, this approach for accurate classification of cells provides a fast, bias-free alternative to manual counting.


Assuntos
Técnicas de Cultura de Células , Azul Tripano , Animais , Humanos , Contagem de Células/métodos , Linhagem Celular , Spodoptera
6.
Vaccine X ; 12: 100223, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36217423

RESUMO

Inactivated polio vaccine production using attenuated Sabin strains (sIPV) instead of wild type polio viruses (cIPV) is an initiative encouraged by the World Health Organization. This use of attenuated viruses is preferred as it reduces risks related to potential outbreaks during IPV production. Previously, an sIPV production process was set up based on the cIPV production process. Optimizing this process while using only animal component free (ACF) substances allows reduction of operational costs and mitigates risks of adverse effects related with animal derived compounds. Here, development of a process for production of sIPV using only ACF compounds, is described. The upstream process required a change in cell growth medium from serum-containing medium to ACF medium, while virus production media remained the same as the already used M199 medium was free of animal components. In the downstream process multiple modifications in existing unit operations were made including addition of a diafiltration step prior to inactivation. After optimizing each unit operation, robustness of the whole process was demonstrated using design of experiments (DoE) methodology. By using DoE we were able to vary different process parameters across unit operations to assess the impact on our quality attributes. The developed process was robust as the observed variation for quality attributes due to differences in process parameters remained within specification. The resulting pilot process showed not only to be robust, but also to have a considerable higher product yield when compared to the serum containing sIPV process. Product yields are now comparable to the cIPV process based on using wild type polio viruses. Moreover, the potency of the produced vaccine was comparable that of cIPV vaccine. The developed ACF sIPV process can be transferred to vaccine manufacturers at the end-of pre-clinical development phase, at lab- or pilot scale, before production of clinical trial material.

7.
Tuberculosis (Edinb) ; 126: 102040, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310626

RESUMO

Two proof of concept clinical trials with TB vaccines demonstrate that new approaches can prevent sustained TB infection in adolescents (BCG revaccination) and TB disease in adults (M72/ASO1E) (Nemes et al., 2018; Tait et al., 2019) [1,2]. Both approaches are in late stage development and provide motivation and rationale to invest into a global TB vaccine pipeline. This pipeline needs to be diverse to address TB-specific challenges including variation in target populations, uncertainties in animal model predictivity and lack of immune correlates of protection. It requires that individual vaccine candidates must be advanced rationally and that the global pipeline must be managed in the most nimble and resource-efficient way, especially in the current constrained funding environment. The TB Vaccine Development Pathway is a webtool which has been developed as an offer to the field to provide a source of information and guidance covering vaccine development from discovery to implementation. It is underpinned by generic and TB vaccine-specific guidelines, regulatory frameworks and best practice, and was compiled by a multi-disciplinary team of scientific and technical experts with the input of the TB vaccine community. The Pathway is a unique tool to guide and accelerate the development of TB vaccine candidates and may be useful for other vaccine development fields.


Assuntos
Desenvolvimento de Medicamentos/tendências , Imunização Secundária/métodos , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose/prevenção & controle , Humanos , Estudos Retrospectivos
8.
Microbiology (Reading) ; 156(Pt 2): 530-542, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19797358

RESUMO

Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations, glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with a pfkA-deficient control strain, which was associated mainly with an increase in CO(2) production, whereas production of by-products was similar in the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated.


Assuntos
Neisseria meningitidis Sorogrupo B/enzimologia , Fosfofrutoquinases/biossíntese , Biomassa , Clonagem Molecular , Escherichia coli/genética , Perfilação da Expressão Gênica , Redes e Vias Metabólicas , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/genética , Fosfofrutoquinases/genética , Filogenia , RNA Bacteriano/genética
9.
Biotechnol Bioeng ; 107(1): 96-104, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20506395

RESUMO

Historical manufacturing data can potentially harbor a wealth of information for process optimization and enhancement of efficiency and robustness. To extract useful data multivariate data analysis (MVDA) using projection methods is often applied. In this contribution, the results obtained from applying MVDA on data from inactivated polio vaccine (IPV) production runs are described. Data from over 50 batches at two different production scales (700-L and 1,500-L) were available. The explorative analysis performed on single unit operations indicated consistent manufacturing. Known outliers (e.g., rejected batches) were identified using principal component analysis (PCA). The source of operational variation was pinpointed to variation of input such as media. Other relevant process parameters were in control and, using this manufacturing data, could not be correlated to product quality attributes. The gained knowledge of the IPV production process, not only from the MVDA, but also from digitalizing the available historical data, has proven to be useful for troubleshooting, understanding limitations of available data and seeing the opportunity for improvements.


Assuntos
Interpretação Estatística de Dados , Previsões , Indústrias/métodos , Modelos Biológicos , Análise Multivariada , Vacina Antipólio de Vírus Inativado/biossíntese , Vacina Antipólio de Vírus Inativado/isolamento & purificação , Animais , Chlorocebus aethiops , Células Vero
10.
Biotechnol Bioeng ; 103(5): 900-8, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19405154

RESUMO

The evolution of vaccine product quality during batch cultivation of Bordetella pertussis, the causative agent of whooping cough, was investigated with the goal to determine the optimal harvest point. The process was explored by measuring mRNA expression at frequent intervals during cultivation. The genes that are involved in virulence are already known for this product and changes in their expression levels are proposed to be indicative for product quality. A quantitative product quality score is calculated based on the expression levels of these virulence genes, which allows comparison of expected product quality between culture samples. Product quality scores were maximal throughout the logarithmic growth phase, but dropped significantly at the start of the stationary phase. This showed that the decreasing lactate and glutamate concentrations towards the end of the batch are critical for product quality. On-line measurement of these nutrients allows the cultivation process to be harvested at the optimal harvest point, increasing process robustness and consistency.


Assuntos
Antígenos de Bactérias/biossíntese , Bordetella pertussis/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Microbiologia Industrial , Vacina contra Coqueluche/normas , Fatores de Virulência/biossíntese , Bordetella pertussis/genética , Meios de Cultura/química , Ácido Glutâmico/análise , Ácido Láctico/análise , Garantia da Qualidade dos Cuidados de Saúde , Fatores de Tempo
11.
Biotechnol Bioeng ; 102(1): 161-7, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18683249

RESUMO

Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter.


Assuntos
Bordetella pertussis/fisiologia , Regulação Bacteriana da Expressão Gênica , Oxigênio/metabolismo , Perfilação da Expressão Gênica , Genes Bacterianos , Análise de Sequência com Séries de Oligonucleotídeos
12.
Bioprocess Biosyst Eng ; 32(3): 289-99, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18668267

RESUMO

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.


Assuntos
Reatores Biológicos/microbiologia , Bordetella pertussis/fisiologia , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Oxigênio/metabolismo , Vacina contra Coqueluche/biossíntese , Coqueluche/prevenção & controle , Algoritmos , Bordetella pertussis/citologia , Proliferação de Células , Simulação por Computador , Retroalimentação/fisiologia , Humanos , Vacina contra Coqueluche/isolamento & purificação
13.
Biotechnol Adv ; 35(5): 565-574, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28522212

RESUMO

Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.


Assuntos
Antígenos/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Bioengenharia , Nanopartículas/química , Antígenos/uso terapêutico , Bactérias Gram-Negativas/genética , Nanopartículas/uso terapêutico , Vesículas Transportadoras/genética
14.
PLoS One ; 11(3): e0150700, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26959983

RESUMO

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.


Assuntos
Vacinas contra Influenza/biossíntese , Vacinas de Produtos Inativados/biossíntese , Difusão Dinâmica da Luz , Eletroforese em Gel de Poliacrilamida , Filtração , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Espectrometria de Massas , Vírion/ultraestrutura
15.
Biotechnol J ; 10(11): 1689-706, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26912077

RESUMO

Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram-negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self-adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV-containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV-producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well-defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.


Assuntos
Proteínas da Membrana Bacteriana Externa , Biotecnologia , Membranas Artificiais , Vacinas , Animais , Humanos , Lipopolissacarídeos , Vacinas Meningocócicas , Camundongos , Nanopartículas , Neisseria meningitidis
16.
Biomed Res Int ; 2015: 358462, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25815312

RESUMO

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus.


Assuntos
Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/virologia , Poliovirus/fisiologia , Replicação Viral , Animais , Células CHO , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Cricetinae , Cricetulus , Humanos , Cinética , Receptores Virais/metabolismo , Tetraspanina 28/metabolismo , Células Vero , Carga Viral
17.
Biotechnol Prog ; 30(6): 1269-76, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25139070

RESUMO

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied. These settings are generally used for cultivation of mammalian cells growing adherent to microcarriers. The rigid disposable vessels showed similar engineering characteristics compared to a traditional glass bioreactor. On the basis of the presented results appropriate settings for adherent cell culture, normally operated at a maximum power input level of 5 W m(-3) , can be selected. Depending on the disposable bioreactor used, a stirrer speed ranging from 38 to 147 rpm will result in such a power input of 5 W m(-3) . This power input will mix the fluid to a degree of 95% in 22 ± 1 s and produce a volumetric mass transfer coefficient of 0.46 ± 0.07 h(-1) .


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Desenho de Equipamento , Modelos Teóricos , Oxigênio/metabolismo
18.
Vaccine ; 32(30): 3721-4, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24801053

RESUMO

Vaccination is the most effective prevention strategy to avoid influenza infection and for protection of large populations. The vast majority of influenza vaccines are still produced with allantoic fluid from fertilized chicken eggs. The presence of ovalbumin, which can constitute over 60% of the total protein content in allantoic fluid, can result in severe allergies. Consequently, efficient reduction of ovalbumin is critical during egg based vaccine manufacturing. Here we present Capto Core 700, a novel core bead chromatographic flow through mode resin for removal of ovalbumin and compare it to sucrose zonal gradient ultracentrifugation, which is the industry standard for egg-based vaccine production. The results demonstrate that core bead chromatography is fully comparable to zonal centrifugation in removing ovalbumin to meet regulatory requirements. Furthermore, the scalability and the shorter process times of this method have the potential to significantly improve the productivity and economy for industrial production compared to zonal centrifugation.


Assuntos
Cromatografia , Orthomyxoviridae/isolamento & purificação , Ovalbumina/química , Óvulo/virologia , Cultura de Vírus/métodos , Animais , Galinhas , Vírus da Influenza A Subtipo H3N2
19.
Vaccine ; 32(24): 2782-8, 2014 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-24583004

RESUMO

Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L(-1)) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1×10(6) cells mL(-1) during batch cultivation to 1.8, 2.7 and 5.0×10(6) cells mL(-1) during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher D-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific D-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific D-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing.


Assuntos
Antígenos Virais/isolamento & purificação , Técnicas de Cultura de Células/métodos , Poliovirus/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Reatores Biológicos , Contagem de Células , Chlorocebus aethiops , Meios de Cultura/química , Células Vero
20.
J Virol Methods ; 193(1): 28-41, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23684847

RESUMO

In order to reduce the time required for the development and production of viral vaccines, host cell lines should be available as expression systems for production of viral vaccines against groups of viral pathogens. A selection of cell lines was compared for their initial feasibility as expression system for the replication of polioviruses, influenza A viruses and respiratory syncytial virus (wild type strain A2). Six adherent cell lines (Vero, HEK-293, MRC-5, CHO-K1, BHK-21 c13, MDCK) and six single cell suspension cell lines (CAP, AGE1.CR.HS, sCHO-K1, BHK-21 c13 2p, MDCK SFS) were studied for their ability to propagate viruses. First, maximum cell densities were determined. Second, virus receptor expression and polarization of the cell lines regarding receptor distribution of eight different viruses were monitored using flow cytometry and immunocytochemistry. Organization of the actin cytoskeleton was studied by transfection of the cells with Lifeact™, a construct coding for actin-EGFP. Finally, the ability to produce virus progeny of the viruses studied was assayed for each cell line. The results suggest that single cell suspension cell lines grown on serum free medium are the best candidates to serve as host cell lines for virus replication.


Assuntos
Tecnologia Farmacêutica/métodos , Vacinas Virais/isolamento & purificação , Animais , Linhagem Celular , Proliferação de Células , Citoesqueleto/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Vírus da Influenza A/crescimento & desenvolvimento , Poliovirus/crescimento & desenvolvimento , Receptores Virais/análise , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Cultura de Vírus/métodos , Replicação Viral
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